Project Summary/Abstract Mitochondrial dysfunction causes mitochondrial diseases and is tightly linked to aging and neurodegenerative disorders such as Parkinson's and Alzheimer's. Recent discoveries support that mitochondrial dysfunction can be overcome to treat age-related decline. Cytegen's hypothesis is that exercise induces the secretion of blood- borne proteins that act systemically to stimulate removal of damaged mitochondria and enrichment of healthy mitochondria (mitochondrial fitness). The company's goal is to identify these proteins to develop into biologics that would serve as a platform to treat the myriad of diseases associated with mitochondrial dysfunction. The discovery platform builds on existing work that exercise reversed mitochondrial dysfunction in a mouse model for mitochondrial disease. Cells derived from the mutant mouse offer an attractive tool for identifying factors that improve mitochondrial fitness. In this application, the feasibility of a cell-based assay platform is tested. This platform measures colony formation, mtDNA copy number, mitochondrial respiration, and expression of mitochondrial quality control proteins, and will be evaluated in Aim 1 using known agonists of mitochondrial biogenesis [bezafibrate and the glitazones (PPAR agonists); metformin and AICAR (AMPK); and resveratrol (Sirt1)]. The results will be integrated to develop a fitness score for each agonist. Whether the assay platform is capable of rapidly differentiating agonists' impact on mitochondrial biogenesis by their fitness score will be determined. The utility of the assay platform for discovery of secreted factors will be evaluated in the second aim. At least one factor identified by Cytegen to be significantly upregulated in exercised, not sedentary, mutant mice will be recombinantly produced and tested in the assay platform. The fitness score will be compared to controls. The completion of this work will identify to what extent the assay platform can be used for discovery purposes. If successful, this platform will help identify substances that enhance mitochondrial fitness that can be further pursued in a Phase II application for ultimate translation into clinical trials.